The term chromatography embraces a family of closely related separation methods. Such methods are all based on the feature that two mutually immiscible phases are brought into contact, wherein one phase is stationary and the other mobile. Chromatography can be used either to purify a liquid from a contaminating compound or to recover one or more product compounds from a liquid.
One area wherein chromatography has recently become of great interest is in the biotechnological field, such as for large-scale economic production of novel drugs and diagnostics. Also, the purification of proteins has recently become of even more importance due to the opening of the field of proteomics, wherein the function of proteins expressed by the human genome is studied. Generally, proteins are produced by cell culture, either intracellularly or secreted into the surrounding medium. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, growth factors, etc. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a sufficient purity, e.g. for use as a human therapeutic, poses a formidable challenge.
Conventionally, cells and/or cell debris has been removed by filtration. Once a clarified solution containing the protein of interest has been obtained, its separation from the other components of the solution is usually performed using a combination of different chromatographic techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, affinity properties, size etc. Several different chromatography matrices are available for each of these techniques, allowing tailoring of the purification scheme to the particular protein involved.
In order to reduce the number of steps required to obtain a product from a cell culture or lysate, improved chromatographic techniques have been presented. For example, an attempt to avoid the step of removing cells and/or cell debris has been made in a technique known as expanded bed chromatography. Expanded bed chromatography is a non-packed bed technique, wherein a matrix, preferably in the form of particles, is brought to a fluidised or expanded state by applying an upward flow of fluid. The solution comprising the compound to be isolated is subsequently introduced into the flow. (For an illustrative use of expanded bed chromatography, see e.g. International patent application WO 98/33572 (Amersham Pharmacia Biotech AB)). It has been shown that in such an expanded bed, cells are in principle allowed to pass through the bed while the desired compound is adsorbed to an appropriate ligand on the particles. However, problems can arise in ion chromatography, in the case where the cell surface and the binding groups of the matrix carry opposite charges. Aggregates are then formed, which may lead to collapse of the expanded bed and thus inducing a decrease of the protein capacity.
An alternative way of improving the available methods for separation of proteins and similar compounds is by improved matrix materials. To this end, matrices that exhibit more than one functionality and hence can adsorb more than one compound selectively have been suggested.
Thus, U.S. Pat. No. 5,522,994 discloses a process for separating molecules of two different sizes from a sample, wherein a separation medium that exhibits at least two different types of functionalities is used. Such a medium can be prepared by treating a porous material having reactive groups within its pores with a modifying agent of a size that penetrates into only certain pores of the porous material. Thus, the modifying agent will then chemically modify the reactive groups only within the pores so penetrated.
WO 98/39094 discloses an alternative use of steric effects in order to provide a separation medium with different properties. In this case, the surface of a porous matrix has been covered with a polymer, which is of such a molecular weight that it cannot penetrate into the micropore system of the matrix. The matrix itself is preferably agarose derivatised with dextran in the pores and optionally functionalised. The polymer that covers the surface can for example also be dextran, however of a larger molecular weight, cellulose or the like. In some cases, the polymer that covers the surface has been functionalised before being attached to the matrix. Thus, the purpose of the polymer surface layer is to sterically prevent compounds that are above a certain size to from passing through towards the inner micropores. By providing the polymer with functionalities that differ from the ones present in the micropore system, a system can be created, wherein smaller compounds that adsorb within the micropores are prevented from adsorbing to the polymer on the surface. The matrices disclosed are useful for isolation of nucleic acids, proteins and other organic and inorganic compounds.
WO 98/39364 describes a process of introducing a second functionality in layers in a porous matrix. This is accomplished by contacting said matrix with a reagent, which reacts with ligands on the matrix surface at a higher reaction rate than it diffuses into the matrix. The reactivity is usually influenced by solvent, pH etc and the diffusion through the matrix will depend upon the chemical nature of matrix and reagent. Accordingly, the process results in a matrix wherein the original ligands are still present within the inner pore system while the added reagent has provided another functionality in an outer layer. The thickness of the outer layer will then be depending on the amount of reagent added. The preferred matrix is agarose, and the reagent can be bromine according to conventional methods.